Mitochondrial oxidative phosphorylation is capable of supplying more than 95% of the total ATP requirement in respiring eucaryotic cells. It is driven by a respiratory chain composed of a number of multimeric membrane proteins that act in series to affect the transfer of electrons from reduced substrates to oxygen. Previous studies have emphasized the importance of the respiratory chain itself in regulating oxidative phosphorylation and have identified cytochrome c oxidase, its terminal member, as a key enzyme in the overall regulation of cellular energy production. At present, it is unclear how eucaryotic cells alter their cytochrome c oxidase activity levels in response to energy demand. However, the recent discovery of isoforms to the nuclearcoded subunits of cytochrome c oxidase in many eucaryotes, including humans, has led to the hypothesis that these polypeptides play a role in the modulation of cytochrome c oxidase activity. In this grant we will address this hypothesis. Initially, we will use the two subunit V isoforms, Va and Vb, of yeast cytochrome c oxidase as a model. Previous studies have shown that these isoforms affect some catalytic properties of holocytochrome c oxidase in vivo and that the expression of their genes, COX5a and COX5b, is differentially regulated by oxygen. Here, we propose to: 1) examine the structural-functional basis for the differential effects of Va and Vb on the electron transport activities of the holoenzyme; 2) determine if Va and Vb alter the proton pumping activity of the holoenzyme; 3) identify the domain(s) in Va and Vb that modulate holoenzyme activities; 4) determine if COX5a and COX5b are oxygen sensors that regulate the number of holocytochrome c oxidase molecules that are assembled in vivo; and 5) develop and use a heterologous complementation system to determine if human cytochrome c oxidase has subunit isoforms that function like yeast Va and Vb. These studies should enhance our understanding of cellular energetics and cytochrome c oxidase structure-function, and may provide an assay as well as a molecular basis for understanding the growing number of human diseases (i.e., tissue specific myopathies, cardiopathies, and hepatopathies) that are being linked to defects in cytochrome c oxidase. In addition, they should provide new opportunities to examine, and possibly modify, the mechanism of cytochrome c oxidase catalysis.